There is a continuing need for a facile and accurate determination of the presence and quantity of biologically-active substances which may be present in fluids at extremely low concentrations. For example, it may be important to determine the presence of exogenous drugs in body fluids, or the presence of various endogenous substances which may be of diagnostic significance. Traditional methods of qualitative and quantitative analysis have been insufficient due to the fact that such substances are present in extremely small amounts, and because small differences in concentration are often of substantial significance.
For example, aminoglycoside antibiotics exhibit high potency and a broad-spectrum anti-bacterial activity against gram-negative and gram-positive organisms. However, these antbiotics have a narrow therapeutic index and are potentially nephrotoxic. The risk of nephrotoxicity is greater in patients with impaired renal function and in those who receive high dosage or prolonged therapy. Consequently, patients treated with aminoglycosides should be under close clinical observation and serum concentrations of aminoglycosides should be monitored to assure adequate levels and to avoid potentially toxic effects. For example, tobramycin treatment of gram-negative infection requires peak serum concentrations of at least 4 mg/L, and concentrations exceeding 10 mg/L are associated with nephrotoxic side effects.
Immunological binding assay methods have undergone evolution from the original competitive binding radioimmunoassay in which a specific radio-isotope-labeled antigen is made to compete with the analyte (antigen) from the test sample for binding to a specific antibody. Generally, the analyte is quantitated by measuring the proportion of radioactivity which becomes associated with the antibody (through attachment of the labeled antigen thereto) to the radioactivity that remains unassociated with the antibody after having physically separated the antibody-bound antigen from the test sample. This proportion is then compared to a standard curve.
Radioimmunoassay techniques are inherently selective, and have enabled the determination of substances which are present in body fluids in amounts as low as 10.sup.-12 to 10.sup.-15 molar. However, the use, production, shipping, handling, and disposal of radioactive material and the maintenance of the required counting equipment has presented substantial disadvantages to its use.
The use of fluorescence labels, attached to either the antibody or the competing ligand, that is, antigen or hapten, offers significant advantages over radioimmunoassay procedures. Such fluorescence assays are based o the assumption that the antibody is unable to distinguish between the labeled substance (antigen) and the unlabeled analyte. Quantitative analysis of the analyte may be accomplished by determining the change in the fluorescence properties of the labeled antigen upon binding to the antibody. If this change in the fluorescence of the bound labeled ligand is sufficiently different from the unbound labeled analyte ligand, it may be used to quantitate the analyte concentration without the separation of the bound labeled antigen from the labeled unbound antigen in the test solution. These assays, based on changes in fluorescence properties upon binding, have employed fluorescence properties such as excitation transfer, polarization, enhancement and quenching.
Due to the fact that separation is not required, these assays have been termed homogeneous assays and examples of this technique are well known in the art. For example, in Analytical Biochemistry, 108 (1980) 156-161, a fluorescein dye pair is employed in a fluorescence excitation transfer immunoassay for morphine. Journal of Immunological Methods, 42, (1981) 93-103 describes a homogeneous substrate-labeled fluorescent immunoassay for IgG in human serum. Clinica Chimica Acta, 73 (1976) 51-55 and J. Clin. Path., 30 (1977) 526-531 describe, respectively, homogeneous polarization and quenching fluoroimmunoassays for the determination of gentamicin levels in body fluids. Further, Clinical Chemistry, Vol. 27, No. 7 (1981) 1190-1197, describes a homogeneous fluorescence polarization immunoassay for monitoring aminoglycoside antibiotics in serum and plasma.
Major limitations of homogeneous fluoroimmunoassays are (1) the presence, in body fluids, of endogenous interferences which generally result from the inherently small signal from the analyte in the presence of a high background emission from endogenous proteins and other species; (2) the generally lower sensitivity limit; and (3) the greater number of liquid transferring steps (pipetting).
Accordingly, non-homogeneous or heterogeneous fluorescence immunoassays are accomplished by the separation of the antibody-bound labeled ligand complex from fluorescing or other substances present in the solution by chemical, physical or immunological differences between the analyte ligand and the antibody-ligand complex. For example, the antibodies may be absorbed or bonded covalently to paper disks, glass or plastic beads or other solid supporting material. When the antibodies are immobilized on the solid support, labeled ligands and the analyte ligand are introduced and allowed to compete for available binding sites on the antibody. The bound labeled fraction is then separated from the labeled ligand and unlabeled ligand remaining in the solution by washing away the unbound labeled and unlabeled ligands from the complex. The labeled ligand-antibody complex can then be measured directly, without removal from the solid phase, by a modified fluorometer, or the fluorescently-labeled ligand can be removed from the solid phase by washing with an appropriate denaturant and measured in the resulting solution in a fluorometer. Alternatively, a ligand attached to a solid support may compete with the analyte ligand for binding with a labeled antibody. The labeled antibody may then be measured after separation and washing to remove the unbound, labeled antibody.
In Clinical Chemistry, Vol. 25, No. 9 (1979), Curry et al. describe a competitive binding fluorescence immunoassay wherein antigen labeled with a fluorescent dye competes with analyte for a limited amount of antibody immobilized on polyacrylamide beads. Clinica Chimica Acta, 78 (1977) 277-284 describes a non-competitive method for the determination of human serum components based upon the fluorescence of a solid phase antibody. Clinica Chimica Acta, 102 (1980) 169-177 details the use of a solid-phase fluorescently-labeled antibody for measurement of serum immunoglobulins. A double antibody fluorescence immunoassay for tobramycin which employs fluorescein-labeled tobramycin, separation and resuspension is described in Clinical Chemistry, Vol. 27, No. 2 (1981) 249-252.
Additional references which describe fluorescence immunoassays are: Clinical Chemistry, Vol. 25, No. 3 (1979) 353-361; Journal of Pharmaceutical Sciences, Vol. 70, No. 5 (1981) 469-475; O'Donnell et al., Analytical Chemistry, Vol 51, p. 33A (1979); and Nakamura et al., Immunoassays in the Clinical laboratory: 211-226 (1979).
While heterogeneous assays have provided advantages over the homogeneous technique due to the separation of the solid-phase antibody-antigen complex, these advantages are ameliorated by the requirement of additional steps in the assay procedure which are not only labor-intensive, but provide an increased possibility of experimental error.
Accordingly, it has been a desideratum to provide a fluoroimmunoassay which would combine the integrated procedure of the separation-free nature of the homogeneous assay with the superior signal-to-noise ratio of the non-homogeneous assay, that is, to conduct a heterogeneous fluoroimmunoassay without the necessity of the separation of the antibody-bound ligands from unbound ligands.
According to the present invention, the presence of an analyte ligand in a liquid phase is determined by fluorescence immunoassay by providing a solid phase supported antibody complexed with fluorescently labeled ligand to a liquid containing the analyte ligand or by providing a solid phase supported antibody and sequentially contacting the solid phase supported antibody first with the analyte ligand and then with the fluorescently labeled ligand, and measuring directly and with great advantage, the fluorometric activity of the fluorescently labeled ligand in the liquid phase without carrying out any separation of the fluorescently labeled ligand bound to the solid phase supported antibody complex.
For example, in one embodiment of the invention, upon mixing fluorescently labeled ligand complexed to solid phase antibody with analyte ligand solution, the analyte ligand undergoes exchange with the solid phase antibody-fluorescently labeled ligand complex, that is, the analyte ligand displaces the fluorescently labeled ligand out of the solid phase antibody-fluorescently labeled ligand complex.
More particularly, in a preferred embodiment, the present invention consists in mixing a solid phase supported antibody-fluorescently labeled antigenic analyte ligand complex with a liquid phase sample containing the antigenic analyte ligand to be determined, the liquid phase sample initially containing no fluorescently labeled antigenic analyte ligand, where the unlabeled antigenic analyte ligand in the liquid phase sample displaces a part of the fluorescently labeled antigenic analyte ligand from the solid phase supported antibody-fluorescently labeled antigenic analyte ligand complex into the liquid phase, resulting in
(1) a solid phase containing: PA1 (2) a liquid phase containing:
(a) solid phase supported antibody fluorescently labeled antigenic analyte ligand, and PA2 (b) solid phase supported antibody-unlabeled antigenic analyte ligand complex, and PA2 (a) fluorescently labeled antigenic analyte ligand displaced from the solid phase supported antibody-fluorescently labeled antigenic analyte ligand complex, and PA2 (b) a reduced concentration of unlabeled antigenic analyte ligand;
measuring the fluorescence of the resulting liquid phase without measuring the fluorescence of the resulting solid phase and without separating the resulting solid phase from the resulting liquid phase; and determining the concentration of the antigenic analyte ligand in the sample corresponding to the measured fluorescence of the resulting liquid phase.
In another embodiment, a highly purified antibody capable of immunologically binding with the analyte ligand is attached to a solid material and mixed with the analyte ligand and a fluorescently-labeled ligand which is capable of binding to the antibody. The fluorescently-labeled ligand and the analyte ligand, if any, are allowed to compete for immunological binding to the antibody attached to the solid material, and the presence of the analyte ligand is assayed by determining the fluorescent activity in the liquid phase without separating any of the bound fluorescent antigen from the solid material.
In another embodiment, the method can be conducted by providing solid-phase supported antibody to a liquid containing analyte ligand to allow analyte ligands to occupy some of the antibody binding sites. Subsequently, a known and fixed amount of fluorescently labeled ligand is introduced to occupy the remaining unoccupied antibody binding sites. The fluorometric activity of the remaining (unbound) fluorescently-labeled ligand is directly and advantageously measured in the liquid phase without separating any of the bound fluorescently labeled ligand from the solid-phase supported antibody.
In another embodiment, the method may be conducted by providing a fluorescently-labeled purified antibody and a ligand attached to a solid material which is capable of immunologically binding with the fluorescently labeled antibody. The solid material supporting the ligand is mixed with the labeled antibody and the analyte, and the solid phase labeled ligand and the analyte are allowed to compete for immunological binding to the antibody. The presence of the analyte ligand is then determined by determining the fluorescent activity of the fluorescently labeled antibody directly in the liquid phase without separating any of the bound fluorescent antibody from the solid material.
While the invention will be exemplified by reference to specific assays for aminoglycoside antibiotics, the invention in general and certain aspects in particular are broad in scope. In general, the invention provides a sensitive method for determining the presence and quantity of a wide range of analytes by measuring the fluorescent activity of fluorescently-labeled ligands. Measurement is made directly in a liquid phase above a solid phase constituted of bound, labeled ligand-antibody complexes without separating the liquid phase from the solid phase. The specific examples hereinafter described regarding the determination of gentamicin and tobramycin are selected to exemplify the invention, which is applicable to the determination of the presence and quantity of any substance which may be assayed by immunological techniques.